The present invention relates to a selection method.
The present invention also relates to an enzyme and a nucleotide sequence coding for same that are useful in a selection method.
In particular, the present invention relates to a method for the selection (e.g. identification and/or separation) of genetically transformed cells and compounds and genetic material for use in the method.
It is well known that when a nucleotide sequence of interest (xe2x80x9cNOIxe2x80x9d) is to be introduced into a population of cells by transformation, only a certain number of the cells are successfully transformed, i.e. receive the NOI. It is then necessary to identify the genetically transformed cells so that these cells may be separated from the non-transformed cells in the population.
A common technique for a selection method includes introducing transformed cells and non-transformed cells into a medium that comprises a substance which the transformed cells are able to tolerate. In that medium the transformed cells are able to survive and grow, while the non-transformed cells are prone to growth inhibition and, in some cases, are killed.
To date, if a population of plant cells has been subjected to genetic transformation, selection of the transformed cells typically takes place using a selection gene which codes for antibiotic resistance or herbicide resistance. The selection gene is coupled to or co-introduced with the NOI to be incorporated into the plant in question, so that both of the two genes are incorporated into some or all of the population of cells.
As not all of the cells may have been transformed, the cells are then cultivated on or in a medium containing the respective antibiotic or herbicide to which the genetically transformed cells are resistant by virtue of the selection gene. In this medium, the transformed cells are able to grow and thus be identified out of the total cell population, since the non-transformed cellsxe2x80x94which do not contain the antibiotic or herbicide resistance gene in questionxe2x80x94have an inhibited growth or even are killed.
These selection methods which rely on the use of antibiotics or herbicides suffer from a number of disadvantages. For example, there is concern amongst some people, such as environmental groups and governmental authorities, as to whether it is environmentally safe to incorporate genes coding for antibiotic resistance and/or herbicide resistance into plants and micro-organisms. This concern is of particular significance for food plants and for micro-organisms which are not designed and/or intended to be used in a closed environment (e.g. micro-organisms for use in agriculture), and also for micro-organisms which are designed for use in a closed environment but which may be released from the closed environment. While these concerns may prove to be unfounded, each concern may nevertheless lead to governmental restrictions on the use of antibiotic resistance genes and/or herbicide resistance genes in e.g. plants.
It is therefore desirable to develop new methods for selecting genetically transformed cells or organisms (or parts thereof) comprising such.
According to a first aspect of the present invention there is provided a selection method for selecting from a population of cells one or more selectable genetically transformed cells, wherein the population of cells comprises selectable genetically transformed cells and possible non-transformed cells; wherein each of the selectable genetically transformed cells comprises a first expressable nucleotide sequence and optionally a second expressable nucleotide sequence; wherein a component or a metabolic derivative thereof when present in a low concentration in a medium is a nutrient for both the selectable genetically transformed cells and the non-transformed cells; wherein the component or the metabolic derivative thereof when present in a high concentration in a medium is toxic to the non-transformed cells; wherein the first nucleotide sequence codes for a gene product capable of converting the component or the metabolic derivative thereof when present in a high concentration in a medium to a nutrient for the selectable genetically transformed cells; the method comprising the step of introducing the population of cells to a medium, wherein the medium optionally comprises a high concentration of the component or the metabolic derivative thereof, and either wherein the component or the metabolic derivative thereof is a source of both carbohydrate and nitrogen for the selectable genetically transformed cells; or wherein if a portion of the component serves as a metabolic substrate and is metabolically converted to a derivatised substrate, then that derivatised substrate is capable of providing an allosteric effect on the gene product.
According to a second aspect of the present invention there is provided a composition comprising a population of cells comprising selectable genetically transformed cells and possible non-transformed cells; and a medium, wherein each of the selectable genetically transformed cells comprises a first expressable nucleotide sequence and optionally a second expressable nucleotide sequence; wherein a component or a metabolic derivative thereof when present in a low concentration in a medium is a nutrient for both the selectable genetically transformed cells and the non-transformed cells; wherein the component or the metabolic derivative thereof when present in a high concentration in a medium is toxic to the non-transformed cells; wherein the first nucleotide sequence codes for a gene product capable of converting the component or the metabolic derivative thereof when present in a high concentration in a medium to a nutrient for the selectable genetically transformed cells; the medium optionally comprising a high concentration of the component or the metabolic derivative thereof, and either wherein the component or the metabolic derivative thereof is a source of both carbohydrate and nitrogen for the selectable genetically transformed cells; or wherein if a portion of the component serves as a metabolic substrate and is metabolically converted to a derivatised substrate, then that derivatised substrate is capable of providing an allosteric effect on the gene product.
According to a third aspect of the present invention there is provided a population of cells comprising selectable genetically transformed cells and possible non-transformed cells; wherein each of the selectable genetically transformed cells comprises a first expressable nucleotide sequence and optionally a second expressable nucleotide sequence; wherein a component or a metabolic derivative thereof when present in a low concentration in a medium is a nutrient for both the selectable genetically transformed cells and the non-transformed cells; wherein the component or the metabolic derivative thereof when present in a high concentration in a medium is toxic to the non-transformed cells; wherein the first nucleotide sequence codes for a gene product capable of converting the component or the metabolic derivative thereof when present in a high concentration in a medium to a nutrient for the selectable genetically transformed cells; and either wherein the component or the metabolic derivative thereof is a source of both carbohydrate and nitrogen for the selectable genetically transformed cells; or wherein if a portion of the component serves as a metabolic substrate and is metabolically converted to a derivatised substrate, then that derivatised substrate is capable of providing an allosteric effect on the gene product.
According to a fourth aspect of the present invention there is provided a selectable genetically transformed cell comprising a first expressable nucleotide sequence and optionally a second expressable nucleotide sequence; wherein a component or a metabolic derivative thereof when present in a low concentration in a medium is a nutrient for the selectable genetically transformed cell and a non-transformed cell; wherein the component or the metabolic derivative thereof when present in a high concentration in a medium is toxic to a non-transformed cell; wherein the first nucleotide sequence codes for a gene product capable of converting the component or the metabolic derivative thereof when present in a high concentration in a medium to a nutrient for the selectable genetically transformed cell; and either wherein the component or the metabolic derivative thereof is a source of both carbohydrate and nitrogen for the selectable genetically transformed cell; or wherein if a portion of the component serves as a metabolic substrate and is metabolically converted to a derivatised substrate, then that derivatised substrate is capable of providing an allosteric effect on the gene product.
According to a fifth aspect of the present invention there is provided a construct for genetically transforming a non-transformed cell to produce a selectable genetically transformed cell; the construct comprising a first expressable nucleotide sequence and optionally a second expressable nucleotide sequence; wherein a component or a metabolic derivative thereof when present in a low concentration in a medium is a nutrient for the selectable genetically transformed cell and a non-transformed cell; wherein the component or the metabolic derivative thereof when present in a high concentration in a medium is toxic to a non-transformed cell; wherein the first nucleotide sequence codes for a gene product capable of converting the component or the metabolic derivative thereof when present in a high concentration in a medium to a nutrient for the selectable genetically transformed cell; and either wherein the component or the metabolic derivative thereof is a source of both carbohydrate and nitrogen for the selectable genetically transformed cell; or wherein if a portion of the component serves as a metabolic substrate and is metabolically converted to a derivatised substrate, then that derivatised substrate is capable of providing an allosteric effect on the gene product.
According to a sixth aspect of the present invention there is provided a vector comprising a construct for genetically transforming a non-transformed cell to produce a selectable genetically transformed cell; the construct comprising a first expressable nucleotide sequence and optionally a second expressable nucleotide sequence; wherein a component or a metabolic derivative thereof when present in a low concentration in a medium is a nutrient for the selectable genetically transformed cell and a non-transformed cell; wherein the component or the metabolic derivative thereof when present in a high concentration in a medium is toxic to a non-transformed cell; wherein the first nucleotide sequence codes for a gene product capable of converting the component or the metabolic derivative thereof when present in a high concentration in a medium to a nutrient for the selectable genetically transformed cell, and either wherein the component or the metabolic derivative thereof is a source of both carbohydrate and nitrogen for the selectable genetically transformed cell; or wherein if a portion of the component serves as a metabolic substrate and is metabolically converted to a derivatised substrate, then that derivatised substrate is capable of providing an allosteric effect on the gene product.
According to a seventh aspect of the present invention there is provided a plasmid comprising a construct for genetically transforming a non-transformed cell to produce a selectable genetically transformed cell; the construct comprising a first expressable nucleotide sequence and optionally a second expressable nucleotide sequence; wherein a component or a metabolic derivative thereof when present in a low concentration in a medium is a nutrient for the selectable genetically transformed cell and a non-transformed cell; wherein the component or the metabolic derivative thereof when present in a high concentration in a medium is toxic to a non-transformed cell; wherein the first nucleotide sequence codes for a gene product capable of converting the component or the metabolic derivative thereof when present in a high concentration in a medium to a nutrient for the selectable genetically transformed cell; and either wherein the component or the metabolic derivative thereof is a source of both carbohydrate and nitrogen for the selectable genetically transformed cell; or wherein if a portion of the component serves as a metabolic substrate and is metabolically converted to a derivatised substrate, then that derivatised substrate is capable of providing an allosteric effect on the gene product.
According to an eighth aspect of the present invention there is provided an organism comprising a selectable genetically transformed cell; wherein the selectable genetically transformed cell comprises a first expressable nucleotide sequence and optionally a second expressable nucleotide sequence; wherein a component or a metabolic derivative thereof when present in a low concentration in a medium is a nutrient for the selectable genetically transformed cell and a non-transformed cell; wherein the component or the metabolic derivative thereof when present in a high concentration in a medium is toxic to a non-transformed cell; wherein the first nucleotide sequence codes for a gene product capable of converting the component or the metabolic derivative thereof when present in a high concentration in a medium to a nutrient for the selectable genetically transformed cell; and either wherein the component or the metabolic derivative thereof is a source of both carbohydrate and nitrogen for the selectable genetically transformed cell; or wherein if a portion of the component serves as a metabolic substrate and is metabolically converted to a derivatised substrate, then that derivatised substrate is capable of providing an allosteric effect on the gene product.
According to a ninth aspect of the present invention there is provided a kit comprising a construct (such as when contained within or on a vector or in a plasmid) for genetically transforming a non-transformed cell to produce a selectable genetically transformed cell; and a medium; the construct comprising a first expressable nucleotide sequence and optionally a second expressable nucleotide sequence; wherein a component or a metabolic derivative thereof when present in a low concentration in a medium is a nutrient for the selectable genetically transformed cell and a non-transformed cell; wherein the component or the metabolic derivative thereof when present in a high concentration in a medium is toxic to a non-transformed cell; wherein the first nucleotide sequence codes for a gene product capable of converting the component or the metabolic derivative thereof when present in a high concentration in a medium to a nutrient for the selectable genetically transformed cell; the medium optionally comprising a high concentration of the component or the metabolic derivative thereof; and either wherein the component or the metabolic derivative thereof is a source of both carbohydrate and nitrogen for the selectable genetically transformed cell; or wherein if a portion of the component serves as a metabolic substrate and is metabolically converted to a derivatised substrate, then that dervatised substrate is capable of providing an allosteric effect on the gene product.
According to a tenth aspect of the present invention there is provided a plant or plant cell prepared from or comprising the above-mentioned aspects of the present invention.
According to an eleventh aspect of the present invention there is provided a plant or plant cell comprising a heterologous enzyme and/or nucleotide sequence encoding same, wherein the heterologous enzyme is glucosamine-6-phosphate deaminase.
This aspect of the present invention is very advantageous. In this regard, not only does the enzyme itself act as a selection means for some transformed cells (for example potato cells) but furthermore it beneficially affects the mobilisation of glycloproteins during conditions of limited nitrogen availability. An example of the latter advantageous aspect is the mobilisation of seed glycoproteins in germinating legumninous seedlings before they have established their symbiotic relationship with micro-organisms (e.g. bacterium) that are capable of fixing atmospheric nitrogen. In this case, the glycoproteins would for example be converted to N-acteyl-glucosamine and then into glucosamine-6-phosphate, which would then be converted to fructose-6-phosphate by the glucosamine-6-phosphate deaminase.
According to a twelfth aspect of the present invention there is provided a foodstuff or food prepared from or comprising the above-mentioned aspects according to the present invention.
In each aspect of the present invention, the metabolic substrate is preferably metabolically converted to a derivatised substrate by the transformed cell.
If component or the metabolic derivative thereof is present in the medium then the component or the metabolic derivative thereof is present in an amount that does not detrimentally affect a major proportion of the transformed cells.
Preferably, if component or the metabolic derivative thereof is present in the medium then the component or the metabolic derivative thereof is present in an amount that does not detrimentally affect substantially most of the transformed cells.
More preferably, if a component or the metabolic derivative thereof is present in the medium then the component or the metabolic derivative thereof is present in an amount that does not detrimentally affect substantially all of the transformed cells.
In one embodiment of the present invention the medium comprises a high concentration of the component or the metabolic derivative thereof.
However, in an alternative embodiment the medium does not necessarily have to comprise a high concentration of the component or the metabolic derivative thereof. In a further aspect, we have even surprisingly found that in some cases the medium need not contain any added quantities of the component or the metabolic derivative according to the present invention. By way of example, this surprising finding was observed in transgenic potato plants according to the present invention wherein those plants comprise cells containing the nagB gene (as discussed herein). In this regard, it is believed that the levels of glucosamine and/or glucosamine-6-phosphate already present (including the endogeneous levels) were sufficiently high as a result of exposure of the plants to the particular culture medium that was used such that the transformed potato cells could metabolise any endogenous glucosamine-6-phosphate whereas in the wild type plants the levels of endogenous glucosamine-6-phosphate were sufficiently toxic so as to destroy their viability.
Thus, other aspects of the present invention include:
The use of glucosamine-6-phosphate deaminase as a selection means for selecting a genetically transformed cell over a non-transformed cell.
The use of a gene coding for glucosamine-6-phosphate deaminase for providing a selection means for selecting a genetically transformed cell over a non-transformed cell.
A gene for providing a selection means for selecting a genetically transformed cell over a non-transformed cell; wherein the gene is obtainable from NCIMB 40852 or NCIMB 40853 or NCIMB 40854.
The use of a gene obtainable from NCIMB 40852 or NCIMB 40853 or NCIMB 40854 for providing a selection means for selecting a genetically transformed cell over a non-transformed cell.
NCIMB 40852 or NCIMB 40853 or NCIMB 40854.
Additional aspects of the present invention include:
A process of inactivating a gene or gene product that is potentially detrimental to a prokaryote when present in the prokaryote by the insertion of at least one intron into the gene (in particular into the coding portion) thereby inactivating the gene or the gene product vis-à-vis the prokaryote.
A prokaryote comprising a gene that would have been potentially detrimental to a prokaryote when present in the prokaryote, but wherein the gene comprises at least one intron which inactivates the gene or the product thereof in the prokaryotexe2x80x94in particular wherein the intron is present within a coding portion of the gene.
With these additional aspects of the present invention, preferably the at least one intron is inserted into a conserved region of the gene, preferably a conserved region within a coding region.
The enzyme glucosamine-6-phosphate deaminase can also be called 2-amino-2-deoxy-D-glucose-6-phosphate ketol isomerase (deaminating). This enzyme has the enzyme commission number EC 5.3.1.10.
In the above aspects, the phrase xe2x80x9c . . . for selecting a genetically transformed cell over a non-transformed cell . . . xe2x80x9d can be alternatively expressed asxe2x80x94for example xe2x80x9c . . . for selecting a genetically transformed cell from one or more non-transformed cells . . . xe2x80x9d.
Thus, according to one aspect of the present invention there is provided a selection system for selecting at least one genetically transformed cell from a population of cells in a medium, wherein the at least one genetically transformed cell is transformed with a nucleotide sequence which encodes a gene product capable of converting a component present in the medium at a level that is toxic to non-transformed cells into a nutrient for the at least one transformed cell; wherein said component provides a source of nitrogen and carbohydrate for the transformed cell and/or wherein said component serves as a metabolic substrate, at least one metabolite of which has an allosteric effect on the gene product. The present invention also provides enzymes and nucleotides useful in that system.
Preferably the component or the metabolic derivative thereof is a source of both carbohydrate and nitrogen for the selectable genetically transformed cell(s); and wherein if a portion of the component serves as a metabolic substrate and is metabolically converted to a derivatised substrate, then that derivatised substrate is capable of providing an allosteric effect on the gene product.
Preferably the component is capable of furnishing to the selectable genetically transformed cell(s) a source of both carbohydrate and nitrogen.
Preferably the component or the metabolic derivative thereof comprises an amine group.
Preferably the component is glucosamine.
Preferably the metabolic derivative of the component that is toxic to the non-transformed cells comprises a phosphate group and/or is formed by phosphate groups that would otherwise be beneficially utilised by a wild type cell. Alternatively or in addition, the metabolic derivative of the component that is toxic to the non-transformed cells is responsible for sequestration of phosphate groups that would otherwise be beneficially utilised by a wild type cell.
Preferably the metabolic derivative of the component that is toxic to the non-transformed cells comprises an amine group.
Preferably the metabolic derivative of the component that is toxic to the non-transformed cells is capable of furnishing to the selectable genetically transformed cell(s) a source of both carbohydrate and nitrogen.
Preferably the metabolic derivative of the component that is toxic to the non-transformed cells is glucosamine-6-phosphate.
Preferably the derivatised substrate comprises an amine group and/or a phosphate group.
Preferably the derivatised substrate is N-acetyl-glucosamine-6-phosphate.
Preferably the first nucleotide sequence comprises an intron.
Preferably the gene product is an enzyme.
Preferably the enzyme is capable of modifying a glycoprotein precursor.
Preferably the enzyme is capable of deaminating a glycoprotein precursor.
Preferably the enzyme is glucosamine-6-phosphate deaminase.
Preferably the enzyme is glucosamine-6-phosphate deaminase obtainable from a micro-organism.
Preferably the enzyme is glucosamine-6-phosphate deaminase obtainable from a bacterium.
Preferably the enzyme is glucosamine-6-phosphate deaminase obtainable from E. Coli. 
Preferably the enzyme glucosamine-6-phosphate deaminase has the amino acid sequence shown as SEQ ID No. 3 or is a variant, homologue or fragment thereof.
Preferably the enzyme glucosamine-6-phosphate deaminase is encoded by either the nucleotide sequence shown as SEQ ID No. 1 or a variant, homologue or fragment thereof or a sequence that is complementary to a sequence that hybridises thereto, or the nucleotide sequence shown as SEQ ID No. 2 or a variant, homologue or fragment thereof or a sequence that is complementary to a sequence that hybridises thereto.
Preferably the glucosamine-6-phosphate deaminase or the gene encoding same is obtainable from NCIMB 40852 or NCIMB 40853 or NCIMB 40854.
Preferably the selectable genetically transformed cell/cells is/are either in vitro within a culture or in vivo within an organism.
Preferably the selectable genetically transformed cell/cells is/are selectable genetically transformed plant cell/cells.
Preferably the second nucleotide sequence is present and wherein the second nucleotide sequence codes for a nucleotide sequence of interest.
Preferably the plant is capable of providing a foodstuff to humans or animals.
Preferably the plant (or part thereof, including cells thereof) is a monocot or a dicot (including legumes).
Preferably the plant (or part thereof, including cells thereof) is any one of guar, potato or maize.
The present invention therefore provides a method for selecting genetically transformed cellsxe2x80x94such as cells into which a nucleotide sequence of interest (xe2x80x9cNOIxe2x80x9d) has been incorporatedxe2x80x94by providing the transformed cells with a selective advantage.
The method of the present invention is not dependent on the preparation of genetically transformed plants containing as a selection means a nucleotide sequence coding for antibiotic or herbicide resistance. Nevertheless, the method of the present invention can be used in conjunction with those earlier selection methods should the need arisexe2x80x94if for example it is desirable to prepare cells that have been or are to be transformed with a number of NOIs.
Also, the selection method of the present invention can be used in conjunction with one or more other known selection methods, such as those that are described in WO 93/05163 (the contents of which are incorporated herein by reference) and/or WO 94/20627 (the contents of which are incorporated herein by reference), should the need arisexe2x80x94if for example it is desirable to prepare cells that have been transformed with a number of NOIs.
In addition, the selection method of the present invention can be used in conjunction with one or more other selection methods according to the present invention should the need arisexe2x80x94if for example it is desirable to prepare cells that have been transformed with a number of NOIs.
A further beneficial use of a combination of selection methods according to the present invention results in a very efficient multiple screening technique. As indicated above, the presence of the component or the metabolic derivative thereof in the medium is an optional feature. Moreover, in some cases, it may not be necessary to add the component or the metabolic derivative thereof to the medium. Both of these aspects of the present invention could be used in a combined selection process that comprises two screening steps. In this regard, and by way of example, the medium in the first screen utilising the selection method of the present invention would not contain added amounts of the component or the metabolic derivative thereof. With this first screen, selectable transformed cells are selected over at least the majority of the non-transformed cells. Then shouldxe2x80x94for examplexe2x80x94any non-transformed cells be accidentally be carried over in that first screen then a second screen can be cried out. In the second screen the selected population of cells are subjected to a second selection method according to the present invention but wherein the component or the metabolic derivative thereof is present in the medium, preferably in a high concentration. In the second screen, only the transformed cells would remain viable.
With this combined aspect of the present invention, the population of cells of the earlier aspects of the present invention can therefore be a pre-selected (e.g. pre-screened) population of cells, wherein the population of cells has been prior selected by one or more selection methods, such as those according to the present invention.
This combined aspect of the present invention can be alternatively expressed as: a selection method for selecting from a population of cells one or more selectable genetically transformed cells, wherein the population of cells comprises selectable genetically transformed cells and possible non-transformed cells; wherein each of the selectable genetically transformed cells comprises a first expressable nucleotide sequence and optionally a second expressable nucleotide sequence; wherein a component or a metabolic derivative thereof when present in a low concentration in a medium is a nutrient for both the selectable genetically transformed cells and the non-transformed cells; wherein the component or the metabolic derivative thereof when present in a high concentration in a medium is toxic to the non-transformed cells; wherein the first nucleotide sequence codes for a gene product capable of converting the component or the metabolic derivative thereof when present in a high concentration in a medium to a nutrient for the selectable genetically transformed cells; and either wherein the component or the metabolic derivative thereof is a source of both carbohydrate and nitrogen for the selectable genetically transformed cells; or wherein if a portion of the component serves as a metabolic substrate and is metabolically converted to a derivatised substrate, then that derivatised substrate is capable of providing an allosteric effect on the gene product; the method comprising the step of introducing the population of cells to a medium and then selecting at least a potion of the transformed cells over the non-transformed cells, and subsequently introducing the at least portion of the transformed cells to a medium that comprises a high concentration of the component or the metabolic derivative thereof.
The present invention also encompasses compositions and kits useful for this combined aspect of the presentxe2x80x94such as the gene or gene product according to the present invention, a first medium containing no component or metabolic derivative thereon and a second medium comprising a high concentration of the component or metabolic derivative thereof.
Furthermore, the selection method of the present invention can be used in conjunction with further selection methods wherein those further selection methods are a combination of one or more other selection methods according to the present invention and one or more known selection methodsxe2x80x94such as those that are dependent on antibiotic or herbicide resistance and/or those that are disclosed in WO 93/05163 and/or WO 94/20627.
In the selection methods of WO 93/05163 and/or WO 94/20627, the manA gene from Escherichia coli, which encodes mannose-6-phosphate isomerase (E.C. 5.3.2.8.), was employed as a selectable marker. This selection marker is suitable for infer alia the transformation of Solanum tuberosum, conferring positive selection in the presence of mannose. In more detail, the coding region of manA was ligated into a CaMV 35S expression cassette, and introduced into a binary vector for plant transformation mediated by Agrobacterium tumefaciens. To allow comparison of kanamycin selection with selection on mannose, the vector also contained a gene for kanamycin resistance, nptII. In order to identify transformants, the construction also contained the B-glucuronidase histochemical marker, uidA. Stable integration of the manA gene was shown by Southern blotting. Extracts from plants transformed with this construct, and selected on mannose, were shown to have specific activities for mannose-6-phosphate isomerase some five hundred fold those of control plants. Expression of manA in transformed cells relieved the metabolic paralysis, usually caused by mannose, while also allowing it to serve as a source of carbohydrate for transformants. These effects combined to impose a stringent selection pressure in favour of transformed cells, which allowed the recovery of transformants with a very low frequency of escapes. The percentage of shoots which were shown to be transgenic after selection on mannose was approximately twice that of shoots selected on kanamycin. The transformants selected on mannose have proven to be stable over three generations of plants propagated from tubers.
Hence, the population of cells of the earlier aspects of the present invention can therefore be a pre-selected (e.g. pre-screened) population of cells, wherein the population of cells has been prior selected by one or more selection methods according to the present invention and/or one or more other selection methods.
In addition, or in the alternative, the transformed cells selected by the selection method of the present invention can be subsequently subjected to one or more selection methods according to the present invention and/or one or more other selection methods.
The present invention also provides an expression system that enables transformed cells to be selected by the selection method of the present invention. The expression system can be expressing or can be capable of expressing the first nucleotide sequence of the present invention. The expression system may be one or more of a vector, construct, plasmid, cell or organism.
If a cell is also to be transformed with a NOI then the expression system will comprise that NOIxe2x80x94which NOI may be present on or in the same vector, construct, plasmid, cell or organism as the first nucleotide sequence. Alternatively the NOI may be present on or in a different vector, construct, plasmid, cell or organism as the first nucleotide sequence. Preferably, the NOI is present on or in the same vector, construct, plasmid, cell or organism as the first nucleotide sequence.
If a cell is to be transformed with one or more NOIs and one or more other genes for one or more other selection methods (such as another selection method according to the present invention and/or a known selection method) those other nucleotide sequences may be present on or in the same vector, construct, plasmid, cell or organism as the first nucleotide sequence. Alternatively one or more of those other nucleotide sequences may be present on or in a different vector, construct, plasmid, cell or organism as the first nucleotide sequence. Preferably, those other nucleotide sequences are present on or in the same vector, construct, plasmid, cell or organism as the first nucleotide sequence. This allows for workers to easily prepare and easily select for cells that have been transformed with a number of NOIs etc.
The term xe2x80x9ccellsxe2x80x9d is intended to refer to any type of cells from which individual genetically transformed cells may be identified and isolated using the method of the invention. Examples of such cells include plant cells, animal cells and micro-organisms such as bacteria, fungi, yeast etc. The term xe2x80x9ccellsxe2x80x9d is also meant to encompass protoplasts, i.e. the protoplasm of a cell enclosed in a membrane but without a cell wall. While it is contemplated that the selection method of the present invention may be used for any type of cell, the method has been found to be particularly suitable for the selection of genetically transformed plant cells.
The term xe2x80x9cpopulation of cellsxe2x80x9d refers to any group of cells which has been subjected to genetic transformation and from which it is desired to identify those cells which have been genetically transformed and to isolate the genetically transformed cells from non-genetically transformed cells. The population may, for example, be a tissue, an organ or a portion thereof, a population of individual cells in or on a substrate, such as a culture of micro-organism cells, for example a population of cells in a solution or suspension, or a whole organism, such as an entire plant.
The term xe2x80x9cselectingxe2x80x9d refers to the process of identifying and/or isolating the genetically transformed cells from the non-genetically transformed cells using the method of the present invention.
The term xe2x80x9ctoxic to the non-transformed cellsxe2x80x9d includes inhibited growth of the non-transformed cells as well as the death thereof.
The term xe2x80x9cmediumxe2x80x9d includes any medium that is capable of providing the transformed cells with a selective advantage, such as a selective growth advantage. For example, the medium may comprise typical ingredients of a growth medium but wherein those ingredients are in such an amount that only the transformed cells are selectively grown. In some embodiments of the present invention, the medium will comprise a component, or a metabolic precursor therefor, according to the present invention, and preferably in a high concentration.
In this regard, that componentxe2x80x94such as an added component or derived from the added precursorxe2x80x94or a metabolic derivative thereof is a nutrient for the non-transformed cells when the component is present in low concentrations and wherein the component or a metabolic derivative thereof is toxic to the non-transformed cells when the component is present in high concentrations.
Preferably, the term xe2x80x9clow concentrationxe2x80x9d means greater than 0 xcexcM to less than 25 xcexcM. Typical preferred examples of low concentrations are in the xcexc-molar range.
Preferably, the term xe2x80x9chigh concentrationxe2x80x9d means at least 25 xcexcM and up to 100 mM (or in some instances even higher). Typical preferred examples of high concentrations are in the milli-molar range.
The term xe2x80x9cnutrientxe2x80x9d includes a substance that is capable of providing directly or indirectly (e.g. via a metabolite thereof) energy or atoms that are beneficially useful for maintenance and/or growth and/or reproduction etc. of the cell, tissue, organ or organism. For example, the term includes a substrate that can be beneficially metabolised and/or beneficially utilised in a metabolic pathway to enable the transformed cells to grow, to proliferate or to be maintained in a viable form.
The term xe2x80x9cgenetically transformedxe2x80x9d includes transformation using recombinant DNA techniques.
The term xe2x80x9cintroducing the population of cells to a mediumxe2x80x9d means adding the population of cells to the medium or vice versa.
If a portion of the component serves as a metabolic substrate and is metabolically converted to a derivatised substrate, wherein that derivatised substrate is capable of providing an allosteric effect on the gene product, then the metabolic conversion can be a one step metabolic conversion process or it can be a multi-step metabolic conversion process.
The component of the present invention may be derived from a metabolic precursor therefor.
The terms xe2x80x9cnon-transformed cellsxe2x80x9d or xe2x80x9cnon-transformed cellxe2x80x9d mean cells or a cell that do not or does not comprise the first nucleotide sequence according to the present invention. The terms also include cells or a cell that do not or does not comprise another first nucleotide sequence when the genetically transformed cells or cell comprise or comprises two different first nucleotide sequences. The terms also include any previously transformed cell but wherein that previously transformed cell does not comprise a first nucleotide sequence according to the present invention or the same number of first nucleotide sequences as the transformed cell or cells.
In a highly preferred embodiment, the first nucleotide sequence is not in its natural environment. In this regard, the first nucleotide sequence may not be native (i.e. foreign) to the cell or organism. In addition, the first nucleotide sequence may be native to the cell or organism but wherein the first nucleotide sequence is operably linked to a promoter that is heterologous to the first nucleotide sequence.
The first nucleotide sequence may be DNA or RNA. Preferably, the first nucleotide sequence is DNA. More preferably, the first nucleotide sequence is recombinant DNA.
Likewise, the second nucleotide sequence may be DNA or RNA. Preferably, the second nucleotide sequence is DNA. More preferably, the second nucleotide sequence is recombinant DNA.
The term xe2x80x9cnucleotide sequence of interestxe2x80x9d (i.e. xe2x80x9cNOIxe2x80x9d) means any desired nucleotide sequence for incorporation into the cells in question to produce genetically transformed cells. Introduction of nucleotide sequences into plants, micro-organisms and animals is widely practised, and it is believed that there are no limitations upon the nucleotide sequences whose presence may be selected (eg. detected) by use of the selection method of the present invention.
By use of the method of the present invention the presence of the NOI in the genetically transformed cells may be determined without the above-mentioned disadvantages associated with the selection systems relying solely on antibiotic resistance and/or herbicide resistance.
The NOI can be any nucleotide sequence of interest, such as any gene of interest. A NOI can be any nucleotide sequence that is either foreign or natural to the cell or organism (e.g. a particular plant) in question. Typical examples of a NOI include genes encoding proteins and enzymes that modify metabolic and catabolic processes. The NOI may code for an agent for introducing or increasing resistance to pathogens. The NOI may even be an antisense construct for modifying the expression of natural transcripts present in the relevant tissues. The NOI may even code for a compound that is of benefit to animals or humans. Examples of NOIs include nucleotide sequences encoding any one or more of pectinases, pectin depolymerases, polygalacturonases, pectate lyases, pectin lyases, rhamno-galacturonases, hemicellulases, endo-xcex2-glucanases, arabinases, or acetyl esterases, or combinations thereof, as well as antisense sequences thereof. The NOI may encode a protein giving nutritional value to a food or crop. Typical examples include plant proteins that can inhibit the formation of anti-nutritive factors and plant proteins that have a more desirable amino acid composition (e.g. a higher lysine content than a non-transgenic plant).
The NOI may even code for an enzyme that can be used in food processing such as chymosin, thaumatin and xcex1-galactosidase. The NOI can be a gene encoding for any one of a pest toxin, an antisense transcript such as that for patatin or xcex1-amylase, ADP-glucose pyrophosphorylase (e.g. see EP-A-0455316), a protease antisense, a glucanase or genomic xcex21,4-endoglucanase.
The NOI may even code for or comprise an intron of a particular gene. Here the intron can be in sense or antisense orientation. In the latter instance, the particular gene could be DNA encoding xcex2-1,4-endoglucanase. Antisense expression of genorric exon or intron sequences as the NOI would mean that the natural xcex2-1,4-endoglucanase expression would be reduced or eliminated but wherein the expression of a xcex2-1,4-endoglucanase gene according to the present invention would not be affected.
The NOI may be the nucleotide sequence coding for the arabinofuranosidase enzyme which is the subject of PCT patent application PCT/EP96/01009 (incorporated herein by reference). The NOI may be any of the nucleotide sequences coding for the ADP-glucose pyrophosphorylase enzymes which are the subject of PCT patent application PCT/EP94/01092 (incorporated herein by reference). The NOI may be any of the nucleotide sequences coding for the xcex1-glucan lyase enzyme which are described in PCT patent application PCT/EP94/03397 (incorporated herein by reference). The NOI may be any of the nucleotide sequences coding for the glucanase enzyme which are described in PCT patent application PCT/EP96/01008 (incorporated herein by reference).
The NOI may also encode a permease or other transport factor which allows the compound or precursor thereof or metabolised derivative thereof to cross the cell membrane and enter the transformed cells. Instead of facilitating uptake of a compound into a cell, the co-introduced nucleotide sequence may alternatively direct the component or precursor thereof or metabolised derivative thereof to a specific compartmentxe2x80x94such as the plasma membrane or into the vacuole or the endoplasmic reticulum.
More than one NOI can be present.
The NOI can be co-introduced with the first nucleotide sequence according to the present invention.
The term xe2x80x9cco-introducedxe2x80x9d means that the two nucleotide sequences may be coupled to each other, or are otherwise introduced together, in such a manner that the presence of the co-introduced first nucleotide sequence in a cell indicates that the NOI has been introduced into the cell, i.e. if the first nucleotide sequence is shown to have been introduced, the probability that the NOI has also been introduced is significantly increased. The two nucleotide sequences may be part of the same genetic construct and may be introduced by the same vector.
The methods described herein may also be used when the co-introduced first nucleotide sequence and the NOI are introduced independently. This may be performed, for example, by using the same bacteria for incorporation of both genes and incorporating a relatively large number of copies of the NOI into the cells, whereby the probability is relatively high that cells which are shown to express the first nucleotide sequence will also contain and express the NOI.
In order for the introduced first nucleotide sequence and optional NOI to be expressed in the transformed cells, the genetic constructs containing the first nucleotide sequence and/or NOI will typically, but not necessarily, contain regulatory sequences enabling expression of the nucleotide sequences, e.g. known promoters and transcription terminators. Thus, the first nucleotide sequence will typically be associated with a promoter, which may be a constitutive or regulatable promoter, and the NOI will typically also be associated with a constitutive or regulatable promoter.
As mentioned above, preferably the gene product of the present invention is the enzyme glucosamine-6-phosphate deaminase (EC 5.3.1.10). A gene coding for glucosamine-6-phosphate deaminase may be obtainable from non-plant organisms, such as E. coli. An example of such a gene is known as the nagB gene. This gene has been cloned and sequenced by Rogers, M J, et al ((1988) Gene 62: 197-207). However, those workers did not suggest the nag B gene could be expressed in plants, let alone be used in a selection method. Alternatively, the glucosamine-6-phosphate deaminase may be obtainable from plants, such as a mung bean shoot. An example of such activity was reported by Veiga, L A ((1968) Plant and Cell Physiol 9: 1-12)xe2x80x94but that author did not report on any sequence information.
The first nucleotide sequence and/or the NOI may comprise one or more introns. In particular, if the first nucleotide sequence and/or the NOI encodes a gene product that can detrimentally affect a bacterium and all or a part (e.g. a plasmid thereof or therein) of that bacterium is used either to propagate the NOI or as a means to transform the cells, then it is highly desirable for that gene product to be inactive in the bacterium. One way of selectively inactivating the gene product in bacteria is to insert one or more introns into the nucleotide sequence of the first nucleotide sequence or the NOI. This intron or those introns would not be removed after transcription in the bacterium but would be so removed in, for example, plants etc.
In a highly preferred embodiment, if the first nucleotide sequence and/or the NOI comprises at least one intron, then that at least one intron is present in a highly conserved region of the first nucleotide sequence or the NOI. Here, the term xe2x80x9cintronxe2x80x9d is used in its normal sense as meaning a nucleotide sequence lying within a coding sequence but being removable therefrom.
We believe that this is the first time that it has been disclosed or suggested that a gene or gene product that is potentially detrimental to a prokaryotexe2x80x94such as a bacteriumxe2x80x94can be inactivated in a prokaryote by the insertion of at least one intron into the gene, especially when the at least one intron is inserted into a conserved region of the gene, more especially when the at least one intron is inserted into a conserved region of a coding region of the gene.
Thus, the present invention also provides a process of inactivating a gene or gene product that is potentially detrimental to a prokaryote when present in the prokaryote by the insertion of at least one intron into the gene thereby inactivating the gene or the gene product vis-à-vis the prokaryote.
Preferably, the present invention also provides a process of inactivating a gene or gene product that is potentially detrimental to a prokaryote when present in the prokaryote by the insertion of at least one intron into the gene thereby inactivating the gene or the gene product, wherein the at least one intron is inserted into a conserved region of the gene.
Alternatively expressed: this aspect of the present invention concerns a process comprising converting a gene or product thereof that is potentially detrimental to a prokaryote to an altered gene or product thereof that is not potentially detrimental to the prokaryote, the process comprising the step of inserting at least one intron into the potentially detrimental gene and in such a manner that the altered gene is formed, preferably wherein the at least one intron is inserted into a conserved region of the gene, more preferably when the at least one intron is inserted into a conserved region of a coding region of the gene.
The present invention also provides a prokaryote comprising a gene that would have been potentially detrimental to a prokaryote when present in the prokaryote, but wherein the gene comprises at least one intron thereby inactivating the gene or the product thereof in the prokaryote.
Preferably, the present invention also provides a prokaryote comprising a gene that would have been potentially detrimental to a prokaryote when present in the prokaryote, but wherein the gene comprises at least one intron thereby inactivating the gene or the product thereof in the prokaryote; and wherein the at least one intron is inserted into a conserved region of the gene, more preferably when the at least one intron is inserted into a conserved region of a coding region of the gene.
The present invention also encompasses products obtainable from the expression of such an altered gene.
In a highly preferred embodiment of this particular aspect of the present invention there is provided a nucleotide sequence shown as SEQ ID No. 2 or a variant, homologue or fragment thereof or a sequence that is complementary thereto. This nucleotide sequence corresponds to the coding region of the nag B gene (see SEQ ID No. 1) but wherein inter alia an intron is present in the sequence. The present invention also covers a construct, vector, plasmid, or transgenic organism (or organ or tissue or cell thereof comprising or expressing this nucleotide sequence. Preferably, the organism is a plant, or cell or tissue thereof.
The present invention also covers the nucleotide sequence shown as SEQ ID No. 2 or a variant, homologue or fragment thereof or a sequence that is complementary thereto when operably linked to and is under the control of a promoter that allows expression of the nucleotide sequence. In this aspect, the promoter may be a cell or tissue specific promoter. If, for example, the organism is a plant then the promoter can be one that affects expression of the nucleotide sequence in any one or more of seed, sterm, sprout, root and leaf tissues.
The term xe2x80x9cpromoterxe2x80x9d is used in the normal sense of the art, e.g. an RNA polymerase binding site in the Jacob-Mond theory of gene expression.
The promoter could additionally include one or more features to ensure or to increase expression in a suitable host. For example, the features can be conserved regions such as a Pribnow Box or a TATA box. The promoters may even contain other sequences to affect (such as to maintain, enhance, decrease) the levels of expression of the nucleotide sequence of the present invention. For example, suitable other sequences include the Shl-intron or an ADH intron. Other sequences include inducible elementsxe2x80x94such as temperature, chemical, light or stress inducible elements.
Also, suitable elements to enhance transcription or translation may be present. An example of the latter element is the TMV 5xe2x80x2 signal sequence (see Sleat Gene 217 [1987] 217-225; and Dawson Plant Mol. Biol. 23 [1993] 97).
The terms xe2x80x9cvariantxe2x80x9d, xe2x80x9chomologuexe2x80x9d or xe2x80x9cfragmentxe2x80x9d in relation to the amino acid sequence for the preferred enzyme of the present invention include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) amino acid from or to the sequence providing the resultant enzyme has glucosamine-6-phosphate deaminase activity, preferably having at least the same activity of the enzyme shown as SEQ ID No. 3. In particular, the term xe2x80x9chomologuexe2x80x9d covers homology with respect to structure and/or function. With respect to sequence homology, preferably there is at least 75%, more preferably at least 85%, more preferably at least 90% homology to the sequence shown as SEQ ID No. 3. More preferably there is at least 95%, more preferably at least 98%, homology to the sequence shown as SEQ ID No. 3.
The terms xe2x80x9cvariantxe2x80x9d, xe2x80x9chomologuexe2x80x9d or xe2x80x9cfragmentxe2x80x9d in relation to the nucleotide sequence coding for the preferred enzyme of the present invention include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the sequence providing the resultant nucleotide sequence codes for or is capable of coding for an enzyme having glucosamine-6-phosphate deaminase activity, preferably having at least the same activity of the enzyme encoded by the sequences shown as SEQ ID No. 1 or SEQ ID No. 2. In particular, the term xe2x80x9chomologuexe2x80x9d covers homology with respect to structure and/or function providing the resultant nucleotide sequence codes for or is capable of coding for an enzyme having glucosamine-6-phosphate deaminase activity. With respect to sequence homology, preferably there is at least 75%, more preferably at least 85%, more preferably at least 90% homology to the sequence shown as SEQ ID No. 1 or SEQ ID No. 2. More preferably there is at least 95%, more preferably at least 98%, homology to the sequence shown as SEQ ID No. 1 or SEQ ID No. 2. However, preferably, a xe2x80x9cvariantxe2x80x9d, xe2x80x9chomologuexe2x80x9d or xe2x80x9cfragmentxe2x80x9d of SEQ ID No. 2 does not include the nucleotide sequence shown as SEQ ID No. 1 on its own.
Preferably, a xe2x80x9cvariantxe2x80x9d, xe2x80x9chomologuexe2x80x9d or xe2x80x9cfragmentxe2x80x9d of SEQ ID No. 2 includes the nucleotide sequence shown as SEQ ID No 1 but wherein at least one intron is present in the sequence.
Preferably, a xe2x80x9cvariantxe2x80x9d, xe2x80x9chomologuexe2x80x9d or xe2x80x9cfragmentxe2x80x9d of SEQ ID No. 2 includes the nucleotide sequence shown as SEQ ID No. 1 but wherein at least one intron is present in a conserved region of the sequence. Preferably, the intron is inserted into a region which encodes a conserved amino acid sequence. Preferably, that conserved amino acid sequence is VVTFNMDEY SEQ ID NO:4.
The terms xe2x80x9cvariantxe2x80x9d, xe2x80x9chomologuexe2x80x9d or xe2x80x9cfragmentxe2x80x9d are synonymous with allelic variations of the sequences.
The term xe2x80x9cvariantxe2x80x9d also encompasses sequences that are complementary to sequences that are capable of hydridising to the nucleotide sequences presented herein. In this respect, preferably the term xe2x80x9cvariantxe2x80x9d encompasses sequences that are complementary to sequences that are capable of hydridising under stringent conditions (e.g. 65xc2x0 C. and 0.1 SSC) to the nucleotide sequences presented herein.
The present invention also covers nucleotide sequences that can hybridise to the nucleotide sequence of the present invention.
The term xe2x80x9chomologyxe2x80x9d as used herein can be equated with the term xe2x80x9cidentityxe2x80x9d. Relative sequence homology (i.e. relative sequence identity) can be determined by commercially available computer programs that can calculate % homology between two or more sequences. A typical example of such a computer program is CLUSTAL.
The term xe2x80x9cvectorxe2x80x9d includes expression vectors and transformation vectors.
The term xe2x80x9cexpression vectorxe2x80x9d means a construct capable of in vivo or in vitro expression.
The term xe2x80x9ctransformation vectorxe2x80x9d means a construct capable of being transferred from one species to anotherxe2x80x94such as from an E.coli plasmid to an Agrobacterium to a plant.
The term xe2x80x9ctissuexe2x80x9d includes tissue per se and organ.
The term xe2x80x9corganismxe2x80x9d in relation to the present invention includes any organism that could comprise the nucleotide sequence coding for the enzyme according to the present invention and/or products obtained therefrom, and/or wherein the nucleotide sequence according to the present invention can be expressed when present in the organism. Preferably the organism is a plant.
The term xe2x80x9ctransgenic organismxe2x80x9d in relation to the present invention includes any organism that comprises the nucleotide sequence coding for the enzyme according to the present invention and/or products obtained therefrom and/or wherein the nucleotide sequence according to the present invention can be expressed within the organism. Preferably the nucleotide sequence is incorporated in the genome of the organism. Preferably the transgenic organism is a plant.
In a highly preferred embodiment, the transgenic organism (or part thereof) does not comprise the combination of a promoter and the nucleotide sequence coding for the enzyme according to the present invention, wherein both the promoter and the nucleotide sequence are native to that organism (or part thereof) and are in their natural environment. Thus, in this highly preferred embodiment the present invention does not cover the native nucleotide coding sequence according to the present invention in its natural environment when it is under the control of its native promoter which is also in its natural environment. In addition, in this highly preferred embodiment, the present invention does not cover the native enzyme according to the present invention when it is in its natural environment and when it has been expressed by its native nucleotide coding sequence which is also in its natural environment and when that nucleotide sequence is under the control of its native promoter which is also in its natural environment. In other words, it is preferred that the nucleotide sequence is heterologous to the organism and/or is under the control of a heterologous promoter.
As mentioned above, the method of the present invention is particularly suitable for the selection of genetically transformed plant cells, thereby allowing identification and isolation of such cells without being essentially dependent on the use of selection genes coding for antibiotic or herbicide resistance.
The selection method of the present invention may be used for selecting cells in vitro. However, the selection method of the present invention may also be employed in vivo in the sense that it is possible to selectively grow transformed organismsxe2x80x94such as plantsxe2x80x94from cells, tissues etc. that comprise the selection system of the present invention.
In vivo use of the selection method of the present invention is of particular importance in connection with genetic transformation performed on whole plants or on plant parts, in which the plants or plant parts comprise both transformed and non-transformed cells, since selection of the transformed cells can, in some instances, be achieved without directly damaging the neighbouring non-transformed cells. For example, in some instances, the transformed cells have a selective advantage compared to the non-transformed cellsxe2x80x94such as the ability to form shootsxe2x80x94but the non-transformed cells do not suffer any severe disadvantage in the sense of being damaged or killed, as is the case with using antibiotics or herbicides.
In certain cases, such as when an improved selection frequency is desired, it may be advantageous for the cells to be transformed with a nucleotide sequence that is a selection gene different to the first nucleotide sequence. This additional, selection nucleotide sequence may be an additional gene coding for an enzyme (or other protein or polypeptide) suitable for selection according to the present invention, or it may be a gene coding for an enzyme (or other protein or polypeptide) for a known selection method, eg coding for resistance to a antibiotic or herbicide or it may be a gene suitable for selection by the selection methods described in WO 93/05163 and/or WO 94/20627. Thus, genetically transformed cells may be selected using a combination of selection techniques. For example, if the transformed cells also possessed genes coding for resistance to at least one antibiotic or herbicide, then the medium could additionally comprise at least one antibiotic or herbicide to which the transformed cells are resistant. In particular, we have found that the medium of the present invention does not impair the effectiveness of the known selection methods that rely on herbicide or antibiotic resistance.
The selective advantage possessed by the transformed cells of the present invention may be any difference or advantage with regard to the non-transformed cells which allows the transformed cells to be readily identified and isolated from the non-transformed cells. This may, for example, be a difference or advantage allowing the transformed cells to be identified by simple visual means, i.e. without the use of a separate assay to determine the presence of a gene that provides the selection means.
As mentioned above, one aspect of the present invention relates to genetically transformed cells which have been selected according to the above method, in particular plant cells, as well as plants, progeny or seeds derived from or derivable from such genetically transformed plant cells. In particular, it is often an advantage that these cells are genetically transformed plant cells whose genome does not contain an introduced (i.e. non-native) nucleotide sequence coding for toxin-resistance, antibiotic-resistance or herbicide-resistance as a selection means. As explained above, there are concerns about whether it is safe to incorporate genes coding for eg antibiotic resistance in eg food plants. Genetically transformed plant cells selected by the method of the present invention which do not contain selection genes for eg antibiotic resistance, as well as plants, progeny and seeds derived from such cells, are therefore clearly advantageous in this respect.
The transformed cells may be prepared by techniques known in the art. For example, if the transformed cells are transformed plant cells reference may be made to EP-B-0470145 and CA-A-2006454.
Even though the selection method according to the present invention is not disclosed in EP-B-0470145 and CA-A-2006454, those two documents do provide some useful background commentary on the types of techniques that may be employed to prepare the transformed plant cells and transgenic plants according to the present invention. Some of these background teachings are now included in the following commentary.
The basic principle in the construction of genetically modified plants is to insert genetic information in the plant genome so as to obtain a stable maintenance of the inserted genetic material.
Several techniques exist for inserting the genetic information, the two main principles being direct introduction of the genetic information and introduction of the genetic information by use of a vector system. A review of the general techniques may be found in articles by Potrykus (Annu Rev Plant Physiol Plant Mol Biol [1991] 42:205-225) and Christou (Agro-Food-Industry Hi-Tech Mar./Apr. 17-27, 1994).
Thus, in one aspect, the present invention relates to a vector system which carries a first nucleotide sequence or construct according to the present invention and which is capable of introducing the nucleotide sequence or construct into the genome of an organism, such as a plant.
The vector system may comprise one vector, but it can comprise at least two vectors. In the case of two vectors, the vector system is normally referred to as a binary vector system. Binary vector systems are described in further detail in Gynheung An et al. (1980), Binary Vectors, Plant Molecular Biology Manual A3, 1-19.
One extensively employed system for transformation of plant cells with a given promoter or nucleotide sequence or construct is based on the use of a Ti plasmid from Agrobacterium tumefaciens or a Ri plasmid from Agrobacterium rhizogenes (An et al. (1986), Plant Physiol. 81, 301-305 and Butcher D. N. et al. (1980), Tissue Culture Methods for Plant Pathologists, eds.: D. S. Ingrams and J. P. Helgeson, 203-208).
Several different Ti and Ri plasmids have been constructed which are suitable for the construction of the plant or plant cell constructs described above.
The first nucleotide sequence or construct of the present invention should preferably be inserted into the Ti-plasmid between the border sequences of the T-DNA or adjacent a T-DNA sequence so as to avoid disruption of the sequences immediately surrounding the T-DNA borders, as at least one of these regions appear to be essential for insertion of modified T-DNA into the plant genome.
As will be understood from the above explanation, if the organism is a plant, then the vector system of the present invention is preferably one which contains the sequences necessary to infect the plant (e.g. the vir region) and at least one border part of a T-DNA sequence, the border part being located on the same vector as the genetic construct. Preferably, the vector system is an Agrobacterium tumefaciens Ti-plasmid or an Agrobacterium rhizogenes Ri-plasmid or a derivative thereof as these plasmids are well-known and widely employed in the construction of transgenic plants, many vector systems exist which are based on these plasmids or derivatives thereof.
In the construction of a transgenic plant the promoter or nucleotide sequence or construct of the present invention may be first constructed in a microorganism in which the vector can replicate and which is easy to manipulate before insertion into the plant. An example of a useful microorganism is E. coli., but other microorganisms having the above properties may be used. When a vector of a vector system as defined above has been constructed in E. coli. it is transferred, if necessary, into a suitable Agrobacterium strain, e.g. Agrobacterium tumefaciens. The Ti-plasmid harbouring the first nucleotide sequence or construct of the invention is thus preferably transferred into a suitable Agrobacterium strain, e.g. A. tumefaciens, so as to obtain an Agrobacterium cell harbouring the promoter or nucleotide sequence or construct of the invention, which DNA is subsequently transferred into the plant cell to be modified.
As reported in CA-A-2006454, a large number of cloning vectors are available which contain a replication system in E. coli and a selection means which allows a selection of the transformed cells. The vectors contain for example pBR322, the pUC series, the M13 mp series, pACYC 184 etc. In this way, the promoter or nucleotide or construct of the present invention can be introduced into a suitable restriction position in the vector. The contained plasmid is used for the transformation in E.coli. The E.coli cells are cultivated in a suitable nutrient medium and then harvested and lysed. The plasmid is then recovered and then analysedxe2x80x94such as by any one or more of the following techniques: sequence analysis, restriction analysis, electrophoresis and further biochemical-molecular biological methods. After each manipulation, the used DNA sequence can be restricted or selectively amplified by PCR techniques and connected with the next DNA sequence. Each sequence can be cloned in the same or different plasmid.
After each introduction method of the first nucleotide sequence or construct according to the present invention in the plants the presence and/or insertion of further DNA sequences may be necessary. If, for example, for the transformation the Ti- or Ri-plasmid of the plant cells is used, at least the right boundary and often however the right and the left boundary of the Ti- and Ri-plasmid T-DNA, as flanking areas of the introduced genes, can be connected. The use of T-DNA for the transformation of plant cells has been intensively studied and is described in EP-A-120516; Hoekema, in: The Binary Plant Vector System Offset-drukkerij Kanters B. B., Alblasserdam, 1985, Chapter V; Fraley, et al., Crit. Rev. Plant Sci., 4:1-46; and An et al., EMBO J. (1985) 4:277-284.
Direct infection of plant tissues by Agrobacterium is a simple technique which has been widely employed and which is described in Butcher D. N. et al. (1980), Tissue Culture Methods for Plant Pathologists, eds.: D. S. Ingrams and J. P. Helgeson, 203-208. For further teachings on this topic see Potrykus (Annu Rev Plant Physiol Plant Mol Biol [1991] 42:205-225) and Christou (Agro-Food-Industry Hi-Tech Mar./Apr. 17-27, 1994). With this technique, infection of a plant may be done on a certain part or tissue of the plant, i.e. on a part of a leaf, a root, a stem or another part of the plant.
Typically, with direct infection of plant tissues by Agrobacterium carrying the first nucleotide sequence or the construct, a plant to be infected is wounded, e.g. by cutting the plant with a razor or puncturing the plant with a needle or rubbing the plant with an abrasive. The wound is then inoculated with the Agrobacterium. The inoculated plant or plant part is then grown on a suitable culture medium.
When plant cells are constructed, these cells are grown and, optionally, maintained in a medium according to the present invention following well-known tissue culturing methodsxe2x80x94such as by culturing the cells in a suitable culture medium supplied with the necessary growth factors such as amino acids, plant hormones, vitamins, etc, but wherein the culture medium comprises a component according to the present invention. Regeneration of the transformed cells into genetically modified plants may be accomplished using known methods for the regeneration of plants from cell or tissue cultures, for example by selecting the transformed shoots and by subculturing the shoots on a medium containing the appropriate nutrients, plant hormones, etc.
Further teachings on plant transformation may be found in EP-A-0449375.
Reference may even be made to Spngstad et al (1995 Plant Cell Tissue Organ Culture 40 pp 1-15) as these authors present a general overview on transgenic plant construction.
In a highly preferred embodiment, the present invention is based on our finding that it is possible to use constructs comprising an expressable gene coding for glucosamine-6-phosphate deaminase to prepare transformed cells wherein the transformed cells can be selected from non-transformed cells.
In addition, the present invention also covers transgenic plants comprising the transformed cells or constructs of the present invention.
Thus, in a highly preferred embodiment the present invention covers transgenic plants comprising transformed cells or constructs that comprise an expressable gene coding for glucosamine-6-phosphate deaminase.
In order to explain in more detail these highly preferred aspects of the present invention, reference shall be made to at least FIGS. 1-11.
In this regard, glucosamine in low concentrations (typically in xcexc-molar concentrations) is metabolised to N-acetyl glucosamine (xe2x80x9cNAGAxe2x80x9d), which in turn is metabolised to N-acetyl glucosamine 6-phosphate (xe2x80x9cNAG6Pxe2x80x9d), which in turn is metabolised to N-acetyl glucosamine 1-phosphate, which in turn is metabolised to UDP-N-acetyl glucosamine. UDP-N-acetyl glucosamine is a useful biological precursor for glycoproteins (Roberts, R M, Plant Physiol, 45: 263-267). This metabolic pathway is schematically shown in FIG. 1. Hence, in low concentrations, glucosamine is a nutrient for plant cells.
However, glucosamine in high concentrations (typically in milli-molar concentrations) is metabolised to glucosamine-6-phosphate (xe2x80x9cGA6Pxe2x80x9d). This is because when glucosamine is supplied in milli-molar concentrations, the levels of this sugar come within the Km range of hexokinase and, in doing so, phosphorylation occurs to form glucosamine 6-phosphate. This metabolic pathway is schematically shown in FIG. 1.
Thus, administration of micro-molar amounts of glucosamine to plant cells leads to formation of NAGA which can be further metabolised; whereas provision of milli-molar amounts of glucosamine leads to an accumulation of GA6P which is undesirable in non-transformed cells (Chen-She, S (1995), New Phytologist 74: 383-392). In this regard, and unlike NAGA, GA6P is not a nutrient for natural plant cells. In fact, GA6P is toxic to natural plant cells. In this regard, the presence of GA6P renders plant cells less hardy. In some instances, the cells may even die as GA6P does not readily enter plant metabolic pathways. Hence, in high concentrations, metabolism of glucosamine produces a metabolite the accumulation of which is toxic for the non-transformed plant cells.
In accordance with the present invention, we then found that GA6P could be enzymatically converted in plant cells to a nutrient. In particular, we found that the enzyme glucosamine-6-phosphate deaminase (such as the enzyme encoded by the nag B gene) could convert GA6P to fructose 6-phosphate (xe2x80x9cF6Pxe2x80x9d) in plant cells. This metabolic pathway is schematically shown in FIG. 2.
As it is well known, F6P is a very beneficial biological substrate as it is a component of the Embden Meyerhof pathway. Hence, in high concentrations, a potentially toxic metabolite of glucosamine can be enzymatically converted to a beneficial nutrient for plant cells. This enzymatic conversion forms the basis of one aspect of the selection method of the present invention.
It is known that the metabolism of glucosamine 6-phosphate by Escherichia coli is facilitated by the enzyme glucosamine 6-phosphate deaminase (EC 5.3.1.10). This enzyme simultaneously catalyses deamination and aldolketose isomerisation to form fructose 6-phosphate (Wolfe, J B and Nakada, H I (1956) Arch Biochem Biophys 64: 489-497. Wolfe J B, et al (1957) Arch Biochem Biophys 66: 333-339). Nevertheless, it has not been suggested before that such an enzyme could be used as a feature of a selection method, let alone be expressed in plant cells.
Of interest, even though the conversion of GA6P to F6P results in the release of NH3xe2x80x94which in high yields is toxic to plantsxe2x80x94we have found that the plant cells are not detrimentally affected. Hence, despite the release of a potentially toxic by-product in the highly preferred selection method of the present invention, that release does not detrimentally affect the overall selection method. This result was highly surprising.
One aspect of the selection method of the present invention provides an additional advantageous feature. In this regard, NAG6P has a positive effect (an allosteric effect) on the conversion of GA6P to F6P by the enzyme glucosamine-6-phosphate deaminase encoded by nagB. This aspect of the present invention is schematically shown in FIG. 3. This surprising finding is in accordance with studies done with E. coli (Calcagno, M, et al (1984) Biochim Biophys Acta 787: 165-173). Thus, should any glucosamine be metabolised to NAGA and in turn eventually to NAG6P then that NAG6P would ensure conversion of GA6P to F6P by the enzyme glucosamine-6-phosphate deaminase or at least aid the conversion step. This is an advantageous feature of the highly preferred selection method of the present invention.
In a preferred aspect of the present invention, an intron is inserted in to the gene encoding glucosamme-6-phosphate deaminase, in particular into a highly conserved region thereof. This modification was done to minimise or to eliminate the detrimental, degradative effect of glucosamine-6-phosphate deaminase on the cell walls of bacteria such as Agrobacterium. As Agrobacterium is often the vector of choice to transform plant cells, it may be necessary to inactivate the glucosamine-6-phosphate deaminase vis-a-vis the bacterium, but not vis-a-vis the plant cells. This inactivation can be achieved by insertion of an intron into the gene coding for the glucosamine-6-phosphate deaminase. In particular, inactivation can be achieved by insertion of an intron into a conserved region of the gene coding for the glucosamine-6-phosphate deaminase.
In this regard, FIG. 4 shows sequences coding for glucosamine-6-phosphate deaminase from Candida albicans (nag 1 gene) SEQ ID NO:5 and glucosamine-6-phosphate deaminase from E. coli (nag B gene) SEQ ID NO:3. In our studies, we chose to insert the intron within the region of the gene that encodes a conserved amino acid sequence, in this case the amino acid sequence VVTFNMDEY SEQ ID NO:4.
Also, FIGS. 5, 6 and 7 schematically present the cloning procedure adopted.
FIGS. 8, 9, 10 and 11 present schematic diagrams of the resultant plasmids.
The following samples were deposited in accordance with the Budapest Treaty at the recognised depositary The National Collections of Industrial and Marine Bacteria Limited (NCIMB) at 23 St. Machar Drive, Aberdeen, Scotland, United Kingdom, AB2 1RY on Jan. 10, 1997:
1. E.coli DH5xcex1 containing plasmid pVictorIV GNG E35S nagB IV2. The deposit number is NCIMB 40852. This plasmid comprises the nagB gene with an intron.
2. E.coli DH5xcex1 containing plasmid pVictorIV GNG rbc nagB IV2. The deposit number is NCIMB 40853. This plasmid comprises the nagB gene with an intron.
3. E.coli DH5xcex1 containing plasmid pVictorIV GNG nagB. The deposit number is NCIMB 40854. This plasmid comprises the nagB gene without an intron.
Highly preferred aspects of the present invention therefore relate to first nucleotide sequences according to the present invention obtainable from those deposits, including expression vectors, constructs, organisms and transgenic organisms comprising those same sequences or plasmids.
The present invention also encompasses a selection means capable of enabling the selection of a transformed cell over a non-transformed cell, wherein the selection means is obtainable from each of those deposits.
For example the present invention encompasses a selection means that is obtainable from deposit number NCIMB 40854. In this regard, the selection means (i.e. the selection means capable of enabling the selection of a transformed cell over a non-transformed cell) may be obtained from this deposit by PCR amplification techniques (such as those mentioned below) and using the following primers:
5xe2x80x2-(B134) (38-mer)
TAAGATCTAAACAACAACATGAGACTGATCCCCCTGAC (SEQ ID NO:6)
3xe2x80x2-(B137) (28-mer)
ACCTCGAGCAGGGATAACAATTACAGAC (SEQ ID NO:7)
By way of further example, the present invention encompasses a selectaion means capable of enabling the selection of a transformed cell over a non-transformed cell, wherein the selection means is obtainable from deposit number NCIMB 40852 or 40853. In this regard, the selection means may be obtained from this deposit by PCR amplification techniques (such as those mentioned below) and using the following primers:
Primer 1 (B507) 29xe2x80x2MER
TGCAGAGATCTAAACAACAACATGAGACT (SEQ ID NO:8)
Primer 6 (B351) 27xe2x80x2MER
CATGCCTCGAGCAGGGATAACAATTAC (SEQ ID NO:9)